Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Biological thresholds of cold-induced phrenic nerve injury.

The effects of controlled cooling on phrenic nerve signal conduction were investigated by cooling an isolated segment of the phrenic nerve with a constant but variable temperature probe. The conduction of a standard electrical stimulus applied to the nerve proximal to the cooled section was measured by detector electrodes sutured to the diaphragm. Nerve conduction of the applied stimulus ceased between 10 degrees and 12 degrees C but returned within seconds after the probe was removed. The delay in the return of conduction increased as nerve temperature decreased until at a temperature of 4 degrees C the ability to conduct did not return after 4 hours. The amount of fat surrounding the nerve and the blood flow rate along the cooled portion of the nerve were observed to ameliorate the effects of low temperature on stimulus conduction. Total body cooling also appears to offer some protection against loss of conduction.